Embryonic stem cells (ES cells) are stem cells established from an inner cell mass of mammalian blastocyst, and can be infinitely grown while maintaining their potential for differentiating into all types of cells (pluripotency). Focusing on this characteristic, there is expectation for stem cell therapy, which comprises treating a patient with myocardial infarction or Parkinson's disease by transplanting myocardial cells or nerve cells produced in large amounts from ES cells. However, ES cells involve the critical ethical issue of utilizing and sacrificing human fertilized eggs. On the other hand, tissue stem cells such as neural stem cells, hematopoietic stem cells, and mesenchymal stem cells are present in individual tissues of a living body. Tissue stem cells do not involve the ethical issue because of the non-use of a fertilized egg, and avoid graft rejection because of the possible use of cells from the patient. However, tissue stem cells are difficult to isolate and the growth potential and differentiation potential thereof are much poorer than those of ES cells. If somatic cells such as tissue stem cells and differentiated cells can be converted to cells similar to ES cells having high growth potential and pluripotency by any means, the resulting ES-like cells would be ideal stem cells for clinical application. Specifically, it is hoped, for example, that somatic cells collected from a patient will be stimulated with a nuclear reprogramming factor (a factor for inducing nuclear reprogramming) to convert to ES-like cells, which ES-like cells will be clinically applied as stem cells. However, there is no system enabling the efficient search of such a nuclear reprogramming factor.
The term ECAT gene (ES cell associated transcript gene) generically refers to a series of genes specifically expressed in totipotent cells such as ES cells. A reported ECAT gene is the transcription factor Oct3 (also called Oct4 or POU5f1; hereinafter referred to as Oct3/4) gene. Although a similar gene has been reported in humans (hOct3/4 gene; Takeda et al., Nucleic Acids Research, 20:4613-4620 (1992)), there is no report of demonstrating the ES-cell-specific expression of the hOct-3/4 gene.
In recent years, our group has found nine genes specifically expressed in ES cells on the basis of computerized analysis utilizing an EST database and Northern blot analysis, and designating them as ECAT1 gene, ECAT2 gene, ECAT3 gene, ECAT4 gene, ECAT5 gene, the ECAT gene 6 gene, ECAT7 gene, ECAT8 gene, and ECAT9 gene (International Patent Publication No. WO 02/097090). Of these, ECAT4 is a factor also called Nanog, and has been shown to be an essential factor for the maintenance of the totipotency (pluripotency) of ES cells (Mitsui, K., et al., Cell, 113: 631-642 (2003)). ECAT5 is a factor also called ERas, and has been shown to promote the growth of ES cells (Takahashi, K., et al., Nature, 423: 541-545 (2003)).
ECAT3 is a kind of F-box-containing protein, namely Fbx15, and is considered to be a ubiquitin ligase because it has the F-box. As a result of an analysis of the expression control region of the ECAT3 gene, ECAT3 was shown to undergo cooperative expression control by the two ES-cell-specific transcription factors Oct4 and Sox2 (Tokuzawa, Y., et al., Molecular and Cellular Biology, 23(8): 2699-2708 (2003)).
As a result of an analysis of a knock-in mouse resulting from knocking in β geo (the fusion gene of the β galactosidase and neomycin resistance genes) to the coding region of the ECAT3 gene, performed to examine the function of ECAT3, no evident abnormalities were observed in the mouse, nor was there any evident abnormality in the growth or differentiation potential of homozygous mutant ES cells. Based on this finding, the ECAT3 gene is considered not to be an essential factor for the maintenance and growth of ES cells (Tokuzawa, Y., et al., Molecular and Cellular Biology, 23(8): 2699-2708 (2003)).